Fucose-branched glycosaminoglycan (fucosylated glycosaminoglycan or fucose-containing glycos-aminoglycan, FGAG), also referred to as fucose-branched chondroitin sulfate (FCS), is a special glycosaminoglycan having sulfated fucose substituents extracted from the body wall or viscera of an echinoderm (Huizeng Fan, et al., Pharmaceutical Journal, 1980, 18(3): 203; Ricardo P. et al., J. Biol. Chem., 1988, 263 (34): 18176; Yutaka K. et al., J. Biol. Clem., 1990, 265:5081).
The existing literature shows that FGAG from sea cucumber have both common properties and differences. First, FGAG from different sources and prepared by different methods have some common features, i.e., FGAG has monosaccharide components including N-acetyl-galactosyl (GalNAc, A, chemical name of said N-acetyl-galactose is N-acetyl-2-deoxy-2-galactosamine, hereafter the same), glucuronosyl (GlcUA, U), fucosyl (Fuc, F) and their sulphate (see above reference of Huizeng Fan, 1980; Ricardo P, 1988; and Ken-ichiro Y. et al., Tetrahedron Letters, 1992, 33(34): 4959). In which, GlcUA and GalNAc (or its sulphate) interconnects through β (1-3) and β (1-4) glycosidic bonds to form a backbone, which is similar to a [GlcUA β(1-3)-GalNAc β(1-4)-] disaccharide repeating structural unit of chondroitin sulfate, while fucose or its sulfate attached to the backbone as a branch.
FGAG from different sources and prepared by different methods have chemical structural differences to varying degrees, for example:
(1) Difference in monosaccharide component ratio. FGAG from different sea cucumber species, from different tissues and even prepared by different methods may have significant difference in monosaccharide components. The monosaccharide components of FGAG from several sources are shown in Table 1.
TABLE 1Monosaccharide components and their sulfate groups of FGAG from several sea cucumberChemical componentsSources(molar ratio)Sea cucumber speciesTissuesA:U:F:—OSO3−ReferencesStichopus Japonicusbody1:1.14:0.97:4.20Huizeng Fan, Pharmaceutical Journal,wall1980, 15: 2671:0.88:0.93:4.01Ken-ichiro Y, Tetrahedron Letters, 1992, 33:49591:1.18:2.78:5.46Yutaka K, J. Biol. Chem., 1990, 265: 50811:0.84:2.38:3.69[b]Yutaka K. Biochem. J., 2002, 132: 335viscera1:1.00:1.00:4.70Huizeng Fan, marine drugs, 1983, (3): 134Stichopus variegatusbody1:1.21:1.29:4.62Judi Chen, Chinese Journal of MarinewallDrugs, 1994, (1): 24Holothuriabody1:0.94:0.84:3.60Huizeng Fan, Pharmaceutical Journal.leucospilotawall1983, 18 (3): 2031:0.96:0.78:1.98Haitang Li, Journal of Chinese MedicinalMaterials, 1999, 22(7): 328Holothuria atrabody1:1.15:0.79:2.7[c]Xiaoli Tang, Journal of Chinese MedicinalwallMaterials, 1999, 22(5): 223Holothuria scabrbody1:1.28:0.68:1.72Jian Chen, Food and FermentationwallIndustries, 2006, 32: 123Ludwigothurea griseabody1:1.17:2.17:2.39[c]Paulo AS, Eur. J. Biochem., 1987, 166: 639wall1:0.90:0.97:2.67[d]Ricardo PV, J. Biol. Chem., 1988, 263:181761:0.92:1.23:2.21Paulo AS, J. Biol. Chem., 1996, 271: 23973[a]expressed as mmol/g in the reference (0.81:0.69:1.93:2.99);[b]expressed as wt % in the reference (16.2:20.3:11.66:23.52);[c]expressed as 0.46:0.54:1.00:1.10 in the reference;[d]expressed as 0.33:0.30:0.32:0.88 in the reference.
The difference between the components of FGAG obtained from different species, tissue origins and different extraction methods mainly lies in the larger change in the ratio of Fuc to sulfate group. It can be judged based on the data that not only different specie sources result in different FGAG, but also different extraction methods may lead to a larger difference in components of the product. For example, Yutaka K al. (1990, 2002), Huizeng Fan et al. (1980) and Ken-ichiro Y et. al (1992) respectively extracted FGAG from the body wall of Stichopus Japonicus, however the molar ratio of fucose in FGAG obtained by the former was 2 to 3 times higher than that of the tatters; while FGAG extracted from the same sea cucumber species (L. grisea) by the same research group also have larger differences in the ratio of fucose component (Paulo A S et al., 1987, 1988). FGAG obtained from the same tissue by different extraction method/time have different ratios of fucose component, it is probably because there exists non-GAG like fucosan pollution in FGAG product with higher ratio of fucose component, or there exists damage and loss of branch groups during the production of FGAG product with lower ratio of fucose component; in addition, it is probably related to the accuracy of content determination method.
(2) Difference in structure of backbone: As there are differences in the position and number of sulfate groups of such as chondroitin sulfate A, C and D, the differences in the position and number of sulfate groups also exist in the backbone of FGAF from different species. For example, the data show that GalNAc on the backbone of FGAG from Stichopus japonicas is sulfated at both 4- and 6-positions (Ken-ichiro Y et al., Tetrahedron Letters, 1992, 33: 4959); GalNAc on the backbone of FGAG from Holothuria leucospilota is sulfated only at 6-position but not 4-position (Huizeng Fan, Pharmaceutical Journal, 1983, 18 (3): 203); and on the backbone of FGAG from L. grisea, about 53% of the GalNAc are 6-sulfated, and small amounts of 4,6-sulfated (about 12%), 4-sulfated (about 4%) and nonsulfated (31%) are found (Lubor Borsig et al. J. Biol. Chem. 2007, 282: 14984).
(3) Difference in fucose residues on branch and their sulfation degree. Data show that FGAG from both S. Japonicus and L. grisea have three types of fucose residue branches, i.e., 2,4-disulfated, 3,4-disulfated, and 4-monosulfated fucoses; and the latter has about 25% of nonsulfated fucose residues, which occur as a cluster at the reducing end of FGAG (Ken-ichiro Y. et al., Tetrahedron Letters, 1992, 33: 4959, Paulo A S. et al., J. Biol. Chem., 1996, 271: 23973).
As shown in Table 1, FGAG from Stichopus japonicus and Stichopus variegates generally show a higher degree of sulfation, while FGAG from L. grisea, Holothuria atra, and Holothuria scabr show lower degree of sulfation.
The existing data shows that FGAG from echinoderm has various biological activities.
Most FGAG from various sources has certain anticoagulant activity (Huizeng Fan, Pharmaceutical Journal, 1980, 15(5): 263; Peiwen Zhang, Chinese Journal of Pharmacology and Toxicology, 1988, 2(2): 98; Paulo A S. et al., J. Biol. Chem. 1996, 271: 23973); however, these natural FGAG also has platelet aggregation-inducing activity (Jia-zeng L. et al, Thromb Haemos, 1988, 54(3): 435; Chunwen Shan, Pharmacology and Clinics of Chinese Materia Medica, 1989, 5(3): 33).
FGAG has also been reported to have biological activities of regulation of blood lipid (His-Hisen L. et al., J. Agic. Food Chem., 2002, 50: 3602), anti-artery atherosclerosis, and inhibition of vascular endothelial proliferation (Tapon-Bretaudiere et al., Thromb. Haemost, 2000, 84: 332; Masahiko I. et al., Atherosclerosis, 1997, 129:27; European patent application, EP 0811635), immunoregulation (Ling Sun et al., Progress in biochemistry and biophysics, 1991, 18 (5): 394; Zuqiong Chen et al., Tianjin Medical Journal, 1987, (5): 278), antineoplastic (Renjie Hu et al., Chinese Journal of Clinical Oncology, 1992, 19(1): 72; Weimin Li et al., Journal of Clinical Oncology, 1985, 12(2): 118) and antivirus (J A. Beutler et al., Antivir. Chem. Chemother., 1993, 4(3), 167; PCT patent application PCT/JP90/00159), and so on.
Research data about antithrombotic activity, anticoagulant activity and action mechanisms and pharmacological targets of FGAG from sea cucumber and its derivatives show that FGAG has an anticoagulant mechanism that is different from that of heparin and dermatan sulfate, the targets of its anticoagulant/antithrombotic effect may relate to:
(1) AT-III: namely, there exists an AT-III-dependent antithrombin activity (Paulo A S et al., J. Biol. Chem., 1996, 271, 23973; Xi Ma, Chinese Journal of Hematology, 1990, 11(5):241); (2) HC-II: namely, there exists an HC-II-dependent antithrombin activity (Hideki Nagase et al., Blood, 1995, 85, (6):1527; Guangsen Zhang, Chinese Journal of Hematology, 1997, 18(3): 127); (3) IIa: namely, inhibiting feedback activation factor XIII of thrombin (IIa) (Nagase H et al., Biochem. J., 1996, 119(1): 63-69); (4) f.xase: inhibiting the activation of factor X by endogenous factor Xase (factor VIII-IX complex) (Hideki Nagase et al., Blood, 1995, 85(6):1527; J P Sheehan et al., Blood, 2006, 107(10): 3876); (5) TFPI: increasing the rate of factor Xa inhibition by TFPI, reducing the inhibitory activity on TF-factor VIIa by TFPI-factor Xa, and stimulating the release of TFPI (Hideki Nagase et al., Thromb Haemost, 1997, 78: 864; T. Bretaudiere et al., Thromb Haemost, 2000, 84: 332); (6) plasmin: promoting plasminogen activation and thus promoting thrombolysis (Xiaoguang Yang et al., Chinese Medical Sciences Journal, 1990, 12(3), 187; Yutaka Kariya et al., Biochem. J., 2002, 132: 335).
Although FGAG from sea cucumber has an important potential application value due to its unique anticoagulant mechanism and good anticoagulant activity intensity, so far FGAG is difficult to be used in clinical and this is mainly because:
(1) FGAG from sea cucumber has both anticoagulant activity and platelet aggregation-inducing activity. In clinical, the aim of anticoagulant is antithrombotic, and antiplatelet is another important approach to anti-thrombosis which is commonly used in clinical. Apparently, in terms of thrombosis, the anticoagulant activity for anti-thrombosis and the platelet-activating effect of promoting thrombosis of FGAG oppose each other, allowing it to be difficult to be used in clinical for preventing and treating hematologic diseases. For example, the studies indicate that in acute disseminated intravascular coagulation model of rabbits, the platelet-activating effect of FGAG from Stichopus japonicas entirely offsets its anticoagulant efficacy (Anguo Li, Journal of Traditional Chinese Medicine University of Hunan, 1991, 11(3): 37).
(2) Administration of pharmacodynamic dose of FGAG from Stichopus japonicas into the blood vessels of living animals can lead to platelet count reduction (Jiazeng Li, Chinese Pharmacological Bulletin, 1985, 6(2): 107). Thrombocytopenia, such as immune heparin-induced thrombocytopenia, may lead to hemorrhagic tendency or may lead to serious or even fatal disseminated intravascular coagulation. Available data show that platelet count reduction induced by FGAG from sea cucumber may be related to its platelet aggregation-inducing activity and thus “withhold” of the platelets in the microvascular (Jiazeng Li et al., Bulletin of Chinese Materia Medic, 1983, 8(5): 35).
(3) It is generally known that a wide range of pharmacological targets are closely related to the side effect of bleeding tendency of anticoagulant, and selective target has become an important evaluation index in the development of new anticoagulant drugs (K A Bauer, Hematology, 2006, (1): 450). FGAG from sea cucumber has different targets from that of the other anticoagulant drugs, however, as mentioned above, its targets are still relatively widespread. Available data show that an anticoagulant dose of FGAG can lead to significant bleeding tendency (Paulo A S et al., British Journal of Haematology, 1998, 101: 647).
Natural FGAG from sea cucumber has unique anticoagulant mechanism and potency, on the other hand has defects that limit its application. So, the acquisition of desired target products through structural modification has become one of the important contents of the application research.
At present, the methods for the chemical structural modification of FGAG comprise peroxide depolymerization (European patent disclosure, EP0408770; Ken-ichiro Y et al., Tetrahedron Letters, 1992, 33: 4959), desulfation or carboxyl reduction (Paulo A S et al., Thrombosis Research, 2001, 102: 167), partial acid hydrolysis (Yutaka Kariya, Biochem. J., 2002, 132: 335; Paulo A S et al., Thrombosis Research, 2001, 102: 167), and etc. These efforts have made some progress. For example, studies show that the platelet aggregation-inducing activity of FGAG from Stichopus japonicas may weaken with the reduction of molecular weight (Huizeng Fan, Journal of Biological Chemistry, 1993, 9(2):146); AT-III-dependent antithrombin activity of the depolymerized product by peroxide of FGAG from Stichopus japonicas may also be reduced (Xi Ma, Chinese Journal of Hematology, 1990, 11(5): 241; Paulo A S et al., J. Biol. Chem. 1996, 271, 23973; Hideki Nagase et al., Blood, 1995, 85 (6):1527); For FGAG from L. grisea, the reduction of molecular weight has a more significant effect on HC-II-dependent antithrombin activity (R G Pacheco et al., Blood Coagulation and Fibrinolysis, 2000, 11:563).
By summarizing the research data about depolymerized product of FGAG (mostly referred to as DHG) from Stichopus japonicas, it is known that it is difficult to obtain anticoagulant active products with desired potency and target feature. For example, the data show that the platelet aggregation-inducing activity may be eliminated until the weight-average molecular weight of FGAG from Stichopus japonicus is reduced to 9000 Da (Huizeng Fan et al., Journal of Biological Chemistry, 1993, 9(2): 146); on the other hand, the anticoagulant activity weakens with the reduction of molecular weight (R G Pacheco et al., Blood Coagulation and Fibrinolysis, 2000, 11:563; Huizeng Fan et al., Journal of Biological Chemistry, 1993, 9(2): 146). According to the DHG-related pharmacological and pharmacodynamic research data, DHG with molecular weight less than 10000 Da was used in early, but in the subsequent more than ten years of research, weight-average molecular weight of DHG is mostly between 12000 and 15000 Da (Hideki Nagase et al., Thromb Haemost, 1997, 77(2): 399; Kazuhisa M et al., Kidney International, 2003, 63: 1548; J P Sheehan et al., Blood, 2006, 107(10): 3876). Apparently, the latter is required to maintain necessary anticoagulant potency, but for FGAG from Stichopus japonicas, the safety of this molecular weight range is in doubt in terms of eliminating platelet-inducing activity and avoiding intravascular administration-induced thrombocytopenia, and such safety has not been reflected and validated in the related research data.
Studies have shown that both hydrolysis and partial desulfation of fucose branches can significantly reduce or abolish anticoagulant activity of FGAG, reduction of carboxyl groups has relatively less effect on anticoagulant activity, but the bleeding tendency is still apparent and the effect on platelet activity is unknown (Paulo A S, J. Biol. Chem., 1996, 271, 23973; Paulo A S et al., British J. Haematology, 1998, 101: 647; Paulo A S et al., Thrombosis Research, 2001, 102: 167).
Research data on chemical and biological activity of FGAG from sea cucumber mainly relates to FGAG from L. grisea and Stichopus japonicas. The extraction method of glycosaminoglycan from Thelenota ananas has been described (Xuexiang Liu et al., Journal of Nanjing University of Traditional Chinese Medicine, 2003, 19 (3): 161), but studies on its structure analysis and biological activity have not been reported.
It is seen from available data that the difference between structures of FGAG from L. grisea and Stichopus japonicas lies in the degree of sulfation. GalNAc backbones have different sulfated types and levels, and fucose branches have substantially the same type; while different types of fucose branches have different compositions and thus have different degrees of sulfation on branches (Ken-ichiro Y et al., Tetrahedron Letters, 1992, 33: 4959; Lubor Borsig et al. J. Biol. Chem. 2007, 282: 14984). According to the relative anticoagulant potency of such two FGAG compared to heparin and/or low molecular weight heparin, it is known that FGAG from Stichopus japonicas has stronger anticoagulant and antithrombotic potency (Norihiko S et al. Thromb Haemost, 1991, 65(4): 369; Paulo A S et al., British J. Haematology, 1998, 101: 647).
However, all the above efforts failed to illuminate the effect of platelet activation and intravenous administration of the obtained product on platelet count, which is the most important factor that limits the application value of FGAG from sea cucumber. Next, although the related documents reported the effect potency of these structure-modified products on certain blood factors (pharmacological targets), but the relationship between structural modification and features of pharmacological action mechanism of the obtained product is not clear. So far, the effect of sulfated position on the activity and action features of FGAG has not been reported. Alteration of sulfated type of the glycosyl groups on backbones and/or branches may obtain a new FGAG having more selective pharmacodynamic characteristics and thus having more application values, however, under the existing conditions, glycosaminoglycan can be treated by nonselective sulfation or defulfation, but it is difficult to be modified by position-selective sulfation or defulfation.
The present inventors find surprisingly by the comparative studies of the chemical structure and biological activity of FGAG from Thelenota ananas that the positions and types of fucose branches of FGAG have an important effect on the biological activity, especially on the platelet aggregation-inducing activity. Thus, FGAG from different species may have remarkable differences in application value. The present inventors also find that different depolymerization degrees and methods have different effects on the strength of biological effect that is produced by FGAG through different targets. Based on such difference, one can obtain the features with special target selectivity, and thus obtain FGAG derivatives for treating and/or preventing specific diseases.
The present inventors first find that fucosylated glycosaminoglycan from Thelenota ananas (THG) has special chemical structure features, and its fucose branch types are different to that of FGAG with known or partially known chemical structure, such as FGAG from L. grisea and Stichopus japonicas; THG also has special biological activities, and its platelet activation action is much lower than that of FGAG from such as Holothuria leucospilota and Stichopus japonicas. 
The present invention demonstrates that THG has an activity of inhibiting endogenous factor X enzyme (f.Xase) and HC-II-dependent antithrombin (f.IIa), and first illuminates the relationship between the depolymerization degree of THG and the potency of the pharmacological action. Based on this, the present invention obtains depolymerized THG (dTHG) with higher ratio of f.Xase inhibition/anti IIa activity (potency ratio). Namely, based on the correlation rule between depolymerization and biological activity, the present invention obtains dTHG product with good anticoagulant potency and special target selectivity, starting from THG with special chemical and biological activity. Said dTHG has no platelet aggregation-inducing activity and does not cause platelet count reduction under conditions of high dose and repeated administration.